Method for imaging and differential analysis of cells

ABSTRACT

Provided are methods for determining and analyzing photometric and morphometric features of small objects, such as cells to, for example, identify different cell states. In particularly, methods are provided for identifying apoptotic cells, and for distinguishing between cells undergoing apoptosis versus necrosis.

RELATED APPLICATIONS

This application is a divisional application of a copending patentapplication Ser. No. 13/302,941, filed on Nov. 22, 2011, which is acontinuation of a patent application Ser. No. 13/191,270, filed on Jul.26, 2011, now issued as U.S. Pat. No. 8,175,371, which is a continuationof patent application Ser. No. 10/593,016, filed on Sep. 14, 2006, nowissued as U.S. Pat. No. 8,103,080, the benefit of the filing dates ofwhich are hereby claimed under 35 U.S.C. §120. Patent application Ser.No. 10/593,016 (U.S. Pat. No. 8,103,080) is a National Stage applicationbased on a prior PCT application, PCT/US05/008870, filed on Mar. 16,2005, the benefit of the filing date of which is hereby claimed under 35U.S.C. §371. PCT/US05/008870 itself is based on a prior provisionalapplication Ser. No. 60/553,502, filed on Mar. 16, 2004, the benefit ofthe filing date of which is hereby claimed under 35 U.S.C. §119(e).

FIELD OF THE INVENTION

The present disclosure relates generally to imaging small moving objectsor particles to analyze and distinguish such objects, and morespecifically, to a system and method for determining and analyzingphotometric and morphometric features of small objects, such as cellsto, for example, identify different cell states.

DESCRIPTION OF THE RELATED ART

Apoptosis is a complex, tightly regulated process by which a cellorchestrates its own destruction in response to specific internal orexternal triggers (Jacobson et al., Cell 88:347, 1997; Rathmell andThompson, Cell 109 (Supp):597, 2002), and proceeds in a manner that isdesigned to prevent damage to surrounding cells and tissues. Apoptoticcells typically appear shrunken, with condensed chromatin and fragmentednuclei. Although plasma membrane integrity is initially preserved, inlater stages the plasma membrane becomes compromised and the cells shedapoptotic bodies consisting of organelles, cytoplasm and/or nuclearfragments. Apoptotic cells are rapidly phagocytosed and eliminated invivo, thus preventing the induction of inflammatory responses, which isa process critical to the maintenance of tissue and immune celldevelopment and homeostasis (Jacobson et al.; Rathmell and Thompson;Vaux and Korsmeyer, Cell 96:245, 1999). Inappropriately low apoptoticrates can result in cancer or autoimmune disease, while high rates canresult in neurodegenerative disease or immunodeficiency (Ashkenazi andDixit, Science 281:1305, 1998; Thompson, Science 267:1456, 1995; Fadeelet al., Leukemia 14:1514, 2000). In contrast, necrotic cell death is alargely unregulated process in which the cells generally have intactnuclei with limited chromatin condensation. Cells undergoing necrosis donot induce an early phagocytic response. Instead, the cells swell andrupture, and the release of cellular contents can result in significantlocal tissue damage and inflammation (Jacobson et al.).

Research aimed at cell death regulation has produced a number of methodsto identify and quantify apoptotic cells, and to distinguish betweencells undergoing apoptosis versus necrosis. Among these, flow cytometryhas become a commonly used tool in the identification and quantificationof apoptosis. Changes in cell size, shape, and granularity associatedwith apoptosis can be inferred from scattered laser light (Ormerod etal., J. Immunol. Methods 153:57, 1992). Early intracellular events, suchas the loss of the mitochondrial inner membrane potential or activationand cleavage of caspases, can also be detected using electro-potentialsensitive dyes (Castedo et al., J. Immunol. Methods 265:39, 2002; Greenand Kroemer, Trends Cell. Biol. 8:267, 1998; Green and Reed, Science281:1309, 1998; Kroemer and Reed, Nat. Med. 6:513, 2000; Lizard et al.,Cytometry 21:275, 1995) or fluorogenic substrates (Komoriya et al., J.Exp. Med. 191:1819, 2000; Smolewski et al., J. Immunol. Methods 265:111,2002; Lecoeur et al., J. Immunol. Methods 265:81, 2002). Another earlyapoptotic event results in exposure of phosphatidylserine on the outersurface of the plasma membrane, which can be detected byfluorochrome-labeled annexin V (van Engeland et al., Cytometry 31:1,1998; Vermes et al., J. Immunol. Methods 184:39, 1995; Koopman et al.,Blood 84:1415, 1994; Verhoven et al., J. Exp. Med. 182:1597, 1995).Apoptotic cells eventually lose the ability to exclude cationicnucleotide-binding dyes and nuclear DNA stains with dyes, such aspropidium iodide and 7-aminoactinomycin D (7-AAD) (Lecoeur et al., 2002;Gaforio et al., Cytometry 49:8, 2002; Ormerod et al., Cytometry 14:595,1993; Schmid et al., J. Immunol. Methods 170:145, 1994; Philpott et al.,Blood 87:2244, 1996). Other techniques that can be used to identityapoptosis include biochemical identification of the activated proteases(e.g., caspases, PARP), release of mitochondrial cytochrome c,quantification of cellular DNA content, and progressive endonucleolyticcleavage of nuclear DNA (Alnemri et al., Cell 87:171, 1996; Kohler etal., J. Immunol. Methods 265:97, 2002; Gong et al., Anal. Biochem.218:314, 1994; Gorczyca et al., Leukemia 7:659, 1993; Gorczyca et al.,Cancer Res. 53:1945, 1993).

As noted above, conventional flow cytometric methods do not providedirect morphologic evidence of cell death. Indeed, these techniquesusually target molecular changes that are associated with apoptosis, butsuch changes are not always specific to apoptosis and may also bepresent in cells undergoing necrotic death (Lecoeur et al., 2002;Lecoeur et al., Cytometry 44:65, 2001; Kerr et al., Br. J. Cancer26:239, 1972). For example, necrotic cells, like advanced (late-stage)apoptotic cells, stain with both annexin V and 7-AAD (Lecoeur et al.,2002; Lecoeur et al., 2001). Thus, visualization of the characteristicmorphologic changes associated with apoptosis is still considered to beabsolutely necessary for its identification (Jacobson et al.;Darzynkiewicz et al. Cytometry 27:1, 1997). Standard microscopictechniques allow visualization of specific molecular and biochemicalchanges associated with apoptosis and also morphologic changes thatdistinguish apoptosis from necrosis. However, these standard techniquesalso require subjective analysis and time-consuming image viewing, whichonly allows for processing of relatively limited numbers of cells and,therefore, makes it difficult to attain statistically valid comparisons(Tarnok and Gerstner, Cytometry 50:133, 2002).

Thus, the need exists for techniques that can provide the statisticalpower offered by flow cytometry coupled with the objective assessmentcapabilities associated with microscopic analysis. For example, interestin the dynamic nature of the living cell and efforts to model cellprocesses (variously termed “cytomics” or “systems biology”) arepowerful drivers for new techniques to acquire ever more comprehensivedata from cells and cell populations.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects and attendant advantages of one or more exemplaryembodiments and modifications thereto will become more readilyappreciated as the same becomes better understood by reference to thefollowing detailed description, when taken in conjunction with theaccompanying drawings, wherein:

FIG. 1 shows a schematic representation of the ImageStream 100™multispectral imaging cytometer.

FIGS. 2A-2C show an analysis of cell death using standard flow cytometryand immunofluorescence microscopy. Untreated Jurkat cells (A), Jurkatcells treated with 1.mu.M CPT for 18 hrs (B), and Jurkat cells treatedwith 0.3% hydrogen peroxide for 1 hour (C) were stained with Alexa Fluor488 conjugated annexin V and 7-AAD. Cells were analyzed either byconventional flow cytometry (using a BD FACSort™) or visualized onslides using a Nikon Eclipse E600 fluorescence microscope equipped withbandpass filters appropriate for Alexa Fluor 488 (535/40 nm) and 7-AAD(630/60 nm) fluorescence. The 2-color dot-plots of annexin V vs. 7-AAD,and the brightfield, combined fluorescence and darkfield microscopicimages are shown.

FIGS. 3A-3E show flow cytometric imaging of untreated, CPT-treated andperoxide-treated Jurkat cells that were stained with Alexa Fluor 488conjugated annexin V and 7-AAD. Peroxide-treated cells were alsoseparately stained with HLA class I-PE. After staining, equal cellnumbers of the three populations of cells were mixed and analyzed by (A)conventional flow cytometry using a FACSort™; and (B) multispectralimaging of cells in flow using an ImageStream 100™ cytometer. The sixchannel images of cells from representative members of the doublepositive (DP), single positive (SP), and double negative (DN)populations identified using the ImageStream 100™ are shown in panels C,D, and E, respectively.

FIGS. 4A-4F show (A) a laser scatter analysis (forward scatter vs. sidescatter) of the “DP gate” population of cells from FIG. 3A, by way ofCellQuest™ software on data obtained from a FACSort™ cytometer; (B) asingle color histogram of HLA class I-PE on DP cells from FIG. 3A asmeasured using a FACSort™ cytometer; (C) backgating of HLA class I-PE⁺cells from FIG. 4B onto the scatter histogram of FIG. 4A, wherein HLAclass I-PE⁺ cells (i.e., peroxide-treated, necrotic) are shown in redand HLA class I-PE⁻ cells (i.e., CPT-treated, apoptotic) are shown inblue; (D) a bivariate plot (scatter histogram) of the “Brightfield Area”vs. the “488 nm Scatter Peak Intensity” produced using IDEAS™ softwareon data obtained using the ImageStream™ cytometer; (E) a single colorhistogram of HLA class I-PE on DP cells from FIG. 3D as measured usingthe ImageStream cytometer; (F) backgating of HLA class I-PE⁺ cells fromFIG. 4E onto the scatter histogram of FIG. 4D, wherein HLA class I-PE⁺cells (i.e., peroxide-treated, necrotic) are shown in red and HLA classI-PE⁻ cells (i.e., CPT-treated, apoptotic) are shown in yellow.

FIG. 5 shows resolution of live, early and late apoptotic, and necroticcells using morphometric features based on scatter intensity,brightfield area, and nuclear area. Backgating of the four cellpopulations that had been identified using alternative criteriaconfirmed their identity as live cells and early apoptotic cells withthe DN and SP cells, respectively, shown in FIG. 3B (shown in blue andgreen, center panel), and as necrotic and late apoptotic cells withcells contained in gates R3 and R4, respectively, shown in FIG. 4 (shownin yellow and red, center panel).

DESCRIPTION

The instant disclosure relates to the use of both photometric andmorphometric features derived from multi-mode imagery of objects (e.g.,cells) in flow to discriminate cell states or types, and cell features,in heterogeneous populations of cells, including both non-adherent andadherent cell types. A surprising result of the instant disclosure isthe ability to discriminate between different cell states, such asdifferentiating and identifying live cells, necrotic cells, and cells inboth the early and late stages of apoptosis, by using uniquecombinations of features provided in the ImageStream™ MultispectralImaging Cytometer and the IDEAS™ data analysis software. Discussed inmore detail below are single-step methods for basic and complexmorphometric classification of objects in flow, which may be combinedwith comprehensive multispectral imagery and photometric features toallow, for example, the identification of different cell features and/orcell types or states not feasible with standard flow cytometry.

In the present description, any concentration range, percentage range,or integer range is to be understood to include the value of any integerwithin the recited range and, when appropriate, fractions thereof (suchas one tenth and one hundredth of an integer, etc.), unless otherwiseindicated. As used herein, the term “about” means ±15%. As used herein,the use of an indefinite article, such as “a” or “an”, should beunderstood to refer to the singular and the plural of a noun or nounphrase (i.e., meaning “one or more” of the enumerated elements orcomponents). The use of the alternative (e.g., “or”) should beunderstood to mean either one, both or any combination thereof of thealternatives.

By way of background, methodologies for simultaneous high speedmultispectral imaging in brightfield, darkfield, and four channels offluorescence of cells in flow were recently developed (see, e.g., U.S.Pat. Nos. 6,211,955 and 6,249,341). FIG. 1 illustrates an exemplaryimaging system (e.g., the ImageStream platform). Cells arehydrodynamically focused into a core stream and orthogonally illuminatedfor both darkfield and fluorescence imaging. The cells aresimultaneously trans-illuminated via a spectrally-limited source (e.g.,filtered white light or a light emitting diode) for brightfield imaging.Light is collected from the cells with an imaging objective lens and isprojected on a charge-coupled detector (CCD). The optical system has anumeric aperture of 0.75 and the CCD pixel size in object space is 0.5microns square, allowing high resolution imaging at event rates ofapproximately 100 cells per second. Each pixel is digitized with 10 bitsof intensity resolution, providing a minimum dynamic range of threedecades per pixel. In practice, the spread of signals over multiplepixels results in an effective dynamic range that typically exceeds fourdecades per image. Additionally, the sensitivity of the CCD can beindependently controlled for each multispectral image, resulting in atotal of approximately six decades of dynamic range across all theimages associated with an object.

Prior to projection on the CCD, the light is passed through a spectraldecomposition optical system that directs different spectral bands todifferent lateral positions across the detector (see, e.g., U.S. Pat.No. 6,249,341). With this technique, an image is optically decomposedinto a set of 6 sub-images, each corresponding to a different colorcomponent and spatially isolated from the remaining sub-images. Thisprocess allows for identification and quantitation of signals within thecell by physically separating on the detector signals that may originatefrom overlapping regions of the cell. Spectral decomposition also allowsmultimode imaging: the simultaneous detection of brightfield, darkfield,and multiple colors of fluorescence, as is exemplified in FIG. 1, whichdepicts a red brightfield illumination source and the associatedtransmitted light images in the red detector channel adjacent tofluorescent and scattered light images in the other spectral channels.The process of spectral decomposition occurs during the image formationprocess rather than via digital image processing of a conventionalcomposite image.

The CCD may be operated using a technique called time-delay-integration(TDI), a specialized detector readout mode that preserves sensitivityand image quality even with fast relative movement between the detectorand the objects being imaged. As with any CCD, image photons areconverted to photocharges in an array of pixels. However, in TDIoperation the photocharges are continuously shifted from pixel to pixeldown the detector, parallel to the axis of flow. If the photochargeshift rate is synchronized with the velocity of the flowing cell'simage, the effect is similar to physically panning a camera: imagestreaking is avoided despite signal integration times that are orders ofmagnitude longer than in conventional flow cytometry. For example, aninstrument may operate at a continuous data rate of approximately 30megapixels per second and integrate signals from each object for 10milliseconds, allowing the detection of even faint fluorescent probeswithin cell images that are acquired at high-speed. Careful attention topump and fluidic system design to achieve highly laminar, non-pulsatileflow eliminates any cell rotation or lateral translation on the timescale of the imaging process (see, e.g., U.S. Pat. No. 6,532,061).

A real-time algorithm analyzes every pixel read from the CCD to detectthe presence of object images and calculate a number of basicmorphometric and photometric features, which can be used as criteria fordata storage. Data files encompassing 10,000-20,000 cells are typicallyabout 100 MB in size and, therefore, can be stored and analyzed usingstandard personal computers. The TDI readout process operatescontinuously without any “dead time,” which means every cell can beimaged and the coincidental imaging of two or more cells at a time, asdepicted in FIG. 1, presents no barrier to data acquisition.

Such an imaging system can be employed to determine morphological,photometric, and spectral characteristics of cells and other objects bymeasuring optical signals, including light scatter, reflection,absorption, fluorescence, phosphorescence, luminescence, etc. As usedherein, morphological parameters may be basic (e.g., nuclear shape) ormay be complex (e.g., identifying cytoplasm size as the differencebetween cell size and nuclear size). For example, morphologicalparameters may include nuclear area, perimeter, texture or spatialfrequency content, centroid position, shape (i.e., round, elliptical,barbell-shaped, etc.), volume, and ratios of any of these parameters.Morphological parameters may also include cytoplasm size, texture orspatial frequency content, volume and the like, of cells. As usedherein, photometric measurements with the aforementioned imaging systemcan enable the determination of nuclear optical density, cytoplasmoptical density, background optical density, and the ratios of any ofthese values. An object being imaged can be stimulated into fluorescenceor phosphorescence to emit light, or may be luminescent, wherein lightis produced without stimulation. In each case, the light from the objectmay be imaged on a TDI detector of the imaging system to determine thepresence and amplitude of the emitted light, the number of discretepositions in a cell or other object from which the light signal(s)originate(s), the relative placement of the signal sources, and thecolor (wavelength or waveband) of the light emitted at each position inthe object.

The present disclosure provides methods of using both photometric andmorphometric features derived from multi-mode imagery of objects inflow. Such methods can be employed as a cell analyzer to determine oneor more cell states or types, and cell features, in heterogeneouspopulations of cells when entrained in a fluid flowing through animaging system. As used herein, cell states or types may include livecells, cells early or late in the process of dying (e.g., apoptoticcells or necrotic cells), cells propagating (e.g., cells in differentphases of division), populations and subpopulations of cells (e.g.,leucocyte subpopulations in blood), etc., and combinations thereof.However, it should also be understood that these exemplary methods mightbe used for imaging and distinguishing other moving objects that haveidentifiable photometric and morphometric features. As used herein,gating refers to a subset of data relating to photometric ormorphometric imaging. For example, a gate may be a numerical orgraphical boundary of a subset of data that can be used to define thecharacteristics of particles to be further analyzed. Here, gates havebeen defined, for example, as a plot boundary that encompasses viable(normal) cells as double negatives (DN gate), or early apoptotic cellsas single positives (SP gate), or late apoptotic and necrotic cells asdouble positives (DP gate). Further, backgating may be a subset of thesubset data. For example, a forward scatter versus a side scatter plotin combination with a histogram from an additional marker (e.g.,HLA-class I-PE) may be used to backgate a subset (e.g., late apoptoticcells) within the initial subset (e.g., late apoptotic and necroticcells).

In using an imaging system as described herein, it should be made clearthat a separate light source is not required to produce an image of theobject (cell), if the object is luminescent (i.e., if the objectproduces light). However, many of the applications of an imaging systemas described herein will require that one or more light sources be usedto provide light that is incident on the object being imaged. A personhaving ordinary skill in the art will know that the location of thelight sources substantially affects the interaction of the incidentlight with the object and the kind of information that can be obtainedfrom the images on a TDI detector.

In addition to imaging an object with the light that is incident on it,a light source can also be used to stimulate emission of light from theobject. For example, a cell having been contacted with probe conjugatedto a fluorochrome (e.g., such as FITC, PE, APC, Cy5, or Cy5.5) willfluoresce when excited by light, producing a correspondingcharacteristic emission spectra from any excited fluorochrome probe thatcan be imaged on a TDI detector. Light sources may alternatively be usedfor causing the excitation of fluorochrome probes on an object, enablinga TDI detector to image fluorescent spots produced by the probes on theTDI detector at different locations as a result of the spectraldispersion of the light from the object that is provided by prism. Thedisposition of these fluorescent spots on the TDI detector surface willdepend upon their emission spectra and their location in the object.

Each light source may produce light that can either be coherent,non-coherent, broadband or narrowband light, depending upon theapplication of the imaging system desired. Thus, a tungsten filamentlight source can be used for applications in which a narrowband lightsource is not required. For applications such as stimulating theemission of fluorescence from probes, narrowband laser light ispreferred, since it also enables a spectrally decomposed, non-distortedimage of the object to be produced from light scattered by the object.This scattered light image will be separately resolved from thefluorescent spots produced on a TDI detector, so long as the emissionspectra of any of the spots are at different wavelengths than thewavelength of the laser light. The light source can be either of thecontinuous wave (CW) or pulsed type, preferably a pulsed laser. If apulsed type illumination source is employed, the extended integrationperiod associated with TDI detection can allow the integration of signalfrom multiple pulses. Furthermore, it is not necessary for the light tobe pulsed in synchronization with the TDI detector.

In the embodiments of the present invention, it is to be understood thatrelative movement exists between the object being imaged and the imagingsystem. In most cases, it will be more convenient to move the objectthan to move the imaging system. However, it is also contemplated thatin some cases, the object may remain stationary and the imaging systemmove relative to it. As a further alternative, both the imaging systemand the object may be in motion, which movement may be in differentdirections and/or at different rates.

In certain aspects, there is provided a method for identifying aspecific cell, comprising directing incident light at a cell, using adetector to obtain a side scatter image, and using the spatial frequencycontent of the side scatter image to identify a specific cell. Withincertain embodiments, the methods of the instant disclosure may be usedto identify a specific cell subpopulation that is part of largerheterogeneous cell population. For example, the methods of thisdisclosure may be used to identify a normal cell, a cell undergoingapoptosis (including early and late stage apoptosis), and a cellundergoing necrosis. Alternatively, the methods of the instantdisclosure may be used to identify cells at particular stages ofreplication (S phase, G phase, M phase, etc.). Thus, in a heterogeneouspopulation of cells, the methods of the invention may be used toidentify at least one apoptotic cell and at least one necrotic cell andat least one normal (viable) cell. In addition, early stage and latestage apoptotic cells may be identified.

In another aspect, the instant disclosure provides a method foridentifying a specific cell, comprising directing incident light at acell, using a detector to obtain a brightfield image, and using thespatial frequency content of the brightfield image to identify aspecific cell. In certain embodiments, the spatial frequency contentanalyzed is of the nucleus. Any of the aforementioned embodiments may beused within the context of this aspect of the invention.

In a further aspect, the instant disclosure provides a method foridentifying a specific cell, comprising contacting a cell with a nuclearmarker, directing incident light at the marked cell, using a detector toobtain an image of the cell, and using the nuclear marker image incombination with the spatial frequency content of the cell image toidentify a specific cell. Again, any of the previous embodiments may beused within this method. In certain embodiments, only a single nuclearmarker is used, such as 7-AAD.

In any of the aforementioned methods, multiple images may be collectedsimultaneously. Furthermore, in any of the aforementioned methods, thereis relative motion between the cell and the detector. In addition, inany of the aforementioned methods, the detector is a time delayintegration charge-coupled detector.

The instant disclosure also provides a kit for use in a multispectralimaging system to identify a specific cell type, comprising a singlenuclear marker, wherein a cell contacted with the single marker for atime sufficient to allow identification of an apoptotic cell or anecrotic cell with the multispectral imaging system, as describedherein.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet, are incorporated herein by reference, intheir entirety. The invention having been described, the followingexamples are intended to illustrate, and not limit, the invention.

EXAMPLES Example 1 Induction of Apoptosis

Human acute T leukemic Jurkat cell line was obtained from ATCC(Rockville, Md.; catalog number CRL-1990) and maintained in RPMI 1640(Gibco, Grand Island, N.Y.) containing 5% fetal bovine serum, 1 mMsodium pyruvate (Mediatech, Herndon, Va.), 100.mu.M nonessential aminoacids, 100 U/ml penicillin, 100.mu.g/ml streptomycin, and 2 mML-glutamine (BioWhittaker, Walkersville, Md.) in 5% CO₂ atmosphere at37° C. The density of exponentially growing cells was less than 3×10⁵cells per ml at the time of all treatments. To induce apoptosis, cellswere treated for 18 hours with 1 μM camptothecin (CPT, Sigma), a DNAtopoisomerase I inhibitor.

Example 2 Induction of Necrosis

Human acute T leukemic Jurkat cell line was obtained from ATCC(Rockville, Md.; catalog number CRL-1990) and maintained in RPMI 1640(Gibco, Grand Island, N.Y.) containing 5% fetal bovine serum, 1 mMsodium pyruvate (Mediatech, Herndon, Va.), 100 μM nonessential aminoacids, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine(BioWhittaker, Walkersville, Md.) in 5% CO₂ atmosphere at 37° C. Thedensity of exponentially growing cells was less than 3×10⁵ cells per mlat the time of all treatments. To induce necrosis, cells were treatedfor 1 hour with 0.3% hydrogen peroxide (Sigma, St. Louis, Mo.).

Example 3 Staining to Identify Apoptotic Cells and Necrotic Cells

Control (untreated) cell, CPT treated (apoptotic) cells, and peroxidetreated (necrotic) cells were independently counted and washed once inphosphate buffered saline (PBS, Fair Lawn, N.J.). Each cell group wasresuspended at 10⁷ cells/ml in annexin V Binding Buffer (BD Pharmingen,San Diego, Calif.) containing Alexa Fluor 488 annexin V (MolecularProbes, Eugene, Oreg.) and 10.mu.M 7-aminoactinomycin D (7-AAD,Molecular Probes) for 10 minutes at room temperature. Necrotic cellswere additionally stained with phycoerythrin (PE)-labeled anti-HLA-A, B,C (clone G46-2.6, BD Pharmingen; anti-HLA class I). Each cell group waswashed in annexin V Binding Buffer, fixed in 2% paraformaldehyde(Sigma), and analyzed as either single populations or as a mixture byflow cytometry and immunofluorescence microscopy.

Example 4 Conventional Flow Cytometry and Imaging Flow Cytometry

For flow cytometry, cell fluorescence data excited by a 488 nm laserwere acquired using the FACSort™ cytometer (BD Immunocytometry Systems,San Jose, Calif.) and analyzed using CellQuest™ (BD ImmunocytometrySystems). For imaging flow cytometry, fixed cells at 5×10⁷ cells per mlwere run at 100 cells per second on an ImageStream100™ (“Beta” version),and the data analyzed using the ImageStream Data Analysis andExploration Software™ (IDEAS™).

Example 5 Instrumentation for Multispectral Imaging Flow Cytometry

FIG. 1 provides an exemplary layout of the ImageStream™ platform. Cellsare hydrodynamically focused into a core stream and orthogonallyilluminated for both darkfield and fluorescence imaging. The cells aresimultaneously trans-illuminated via a spectrally-limited source (e.g.,filtered white light or a light emitting diode) for brightfield imaging.Light is collected from the cells with an imaging objective lens and isprojected on a charge-coupled detector (CCD). The optical system has anumeric aperture of 0.75 and the CCD pixel size in object space is 0.5microns square, allowing high resolution imaging at event rates ofapproximately 100 cells per second. Each pixel is digitized with 10 bitsof intensity resolution, providing a minimum dynamic range of threedecades per pixel. In practice, the spread of signals over multiplepixels results in an effective dynamic range that typically exceeds fourdecades per image. Additionally, the sensitivity of the CCD can beindependently controlled for each multispectral image, resulting in atotal of approximately six decades of dynamic range across all theimages associated with an object.

Prior to projection on the CCD, the light is passed through a spectraldecomposition optical system that directs different spectral bands todifferent lateral positions across the detector (see, e.g., U.S. Pat.No. 6,249,341). With this technique, an image is optically decomposedinto a set of 6 sub-images, each corresponding to a different colorcomponent and spatially isolated from the remaining sub-images. This isexemplified in FIG. 1, which depicts a red brightfield illuminationsource and the associated transmitted light images in the red detectorchannel adjacent to fluorescent and scattered light images in the otherspectral channels. The process of spectral decomposition occurs duringthe image formation process rather than via digital image processing ofa conventional composite image.

The CCD is operated using time-delay-integration (TDI), in which imagephotons converted to photocharges in an array of pixels are continuouslyshifted (at a rate synchronized with the velocity of the flowing cell'simage) from pixel to pixel down the detector and parallel to the axis offlow to avoid image streaking. For example, the instrument can operateat a continuous data rate of approximately 30 megapixels per second andintegrate signal from each object for 10 milliseconds, which allows thedetection of even faint fluorescent probes within cell images that areacquired at high speed. Attention to pump and fluidic system design toachieve highly laminar, non-pulsatile flow can eliminate cell rotationor lateral translation on the time scale of the imaging process (see,e.g., U.S. Pat. No. 6,532,061). Every pixel read from the CCD isanalyzed by a real-time algorithm that detects the presence of objectimages and calculates a number of basic morphometric and photometricfeatures, which can be used as criteria for data storage. Data filesencompassing 10,000-20,000 cells can be about 100 MB in size, and arestored and analyzed using standard personal computers.

Example 6 Immunofluorescence Microscopy

Fixed control and treated cells were placed on a conventional glassslide (Erie Scientific, Portsmouth, N.H.), mixed 1:1 with Antifade(Molecular Probes) and covered with a cover slip. The cells werevisualized at 400× using an Eclipse E600 (Nikon, Melville, N.Y.)fluorescence microscope equipped with filters appropriate for AlexaFluor 488 (535/40 nm emission) and 7-AAD (630/60 nm emission).

Example 7 Conventional Analysis of Cells Induced to Undergo Apoptosis orNecrosis

Jurkat T cells were treated with peroxide (to induce necrosis), CPT (toinduce apoptosis, which contained cells in both early and late stages ofapoptosis), or were untreated (control). The three cell populations werethen stained with Alexa Fluor 488 annexin V and 7-AAD and evaluated bybrightfield, darkfield, and fluorescence microscopy, and by conventionalflow cytometry (FIG. 2). The vast majority (>98%) of the control cellswere viable at the time of staining, and were annexin V⁻, 7-AAD⁻ (doublenegative, DN; FIG. 2A). CPT-treated (apoptotic) cells had twopopulations of cells, those that were annexin V⁺ (single positive, SP,or early apoptotic cells), and those that were annexin V⁺, 7-AAD⁺(double positive, DP, or late apoptotic cells) (FIG. 2B). Similar tolate apoptotic cells, peroxide-treated (necrotic cells) also stainedpositively with both annexin V and 7-AAD (FIG. 2C). However, thecondensed, fragmented nuclei of late apoptotic cells could be easilydistinguished from the intact nuclei of necrotic cells byimmunofluorescence microscopy. In addition, apoptotic cells exhibitedgreater darkfield intensity and texture as compared to necrotic cells(see B and C image panels on right, respectively).

Example 8 Analysis of Heterogeneous Cell Population Normal, Apoptoticand Necrotic

A mixture of control, apoptotic, and necrotic Jurkat cells (individuallyprepared as described in Examples 1 and 2) were analyzed in parallel byconventional flow cytometry and on an ImageStream 100™ (Beta system,multispectral imaging flow cytometer). In this experiment, all cellswere stained with Alexa Fluor 488-conjugated annexin V and 7-AAD.Necrotic cells were also stained with PE-conjugated anti-HLA class Ibefore mixing the cell populations to aid in distinguishing necroticcells from late stage apoptotic cells, and to permit “backgating” whennecessary. On the ImageStream™, each cell was simultaneously imaged indarkfield (488 nm laser side scatter), green fluorescence (500-550 nm,annexin V channel), orange fluorescence (550-600 nm, PE channel), redfluorescence (600-650 nm, 7-AAD channel), and brightfield (660-720 nm).Cells were grouped into live (DN), early apoptotic (SP), or doublepositive (DP, which would include late apoptotic and necrotic cells)populations based on the total intensities of annexin V and 7-AADstaining.

Similar bivariate dot-plots of annexin V and 7-AAD staining wereobtained in analyses from both the conventional flow cytometer (see FIG.3A) and the multispectral imaging flow cytometer (see FIG. 3B). However,a unique aspect of data collected on the ImageStream™ is that each datapoint can be “clicked on” to observe the cell imagery associated witheach data point. Consequently, each population gate can be used toperform a “virtual cell sort” by displaying individual images of cellsthat fall within each gate—for example, representative images of cellscontained in the DP, SP and DN gates can be “virtually sorted,” as shownin FIGS. 3C, 3D and 3E, respectively, with each image row representing adifferent cell. Early apoptotic cells (cells in the SP gate) appearslightly shrunken, with more complex brightfield and darkfieldmorphologies, as compared to live cells in the DN gate. The doublepositive (DP) population contains cells with two distinctmorphologies—one containing small, irregularly shaped cells withcondensed, fragmented nuclei; and a second containing larger cells withlarge, unfragmented nuclei that stained uniformly with 7-AAD. Themorphology of these two populations of cells is consistent with cells inthe late stage of apoptosis and necrosis, respectively. Thus, in theabsence of imagery provided by the multispectral imaging flow cytometer,data obtained from a conventional flow cytometer does not permitdiscrimination of similarly stained cells, such as late apoptotic cellsfrom necrotic cells.

Example 9 Conventional Methods to Distinguish Late Apoptotic andNecrotic Cells

As noted in Example 8, although advanced apoptotic and necrotic cellsdiffer morphologically, they cannot be distinguished based solely onannexin V and 7-AAD fluorescence. Plotting the mixed late apoptotic andnecrotic DP population on a forward scatter vs. side scatter (FSC vs.SSC) plot reveals two distinct populations of cells (see FIG. 4A).Analysis of the DP population of cells obtained on the conventional flowcytometer for staining with PE (which was used to stain only thenecrotic subpopulation of cells) permits separation of the necrotic andapoptotic subpopulations of cells (FIG. 4B). Backgating the PE positivenecrotic population in blue reveals that the low SSC population consistsof necrotic cells (FIG. 4C). However, without the aid of an extra marker(in this case, anti-HLA class I-PE) or imagery as described in Example8, data obtained from a conventional flow cytometer does not permitdiscrimination of similarly stained cells, such as late apoptotic cellsfrom necrotic cells.

Example 10 Multispectral Identification of Late Apoptotic and NecroticCells

Analysis of the DP population with IDEAS™ software for size (BrightfieldArea) and Scatter Peak Intensity also revealed two populations of cells(FIG. 4D). The nuclei of cells that fell within the high brightfieldarea, low scatter peak intensity area (R3) were intact, uniformlystained with 7-AAD, and had a morphology consistent with necrotic cells.The nuclei of cells that fell within the low brightfield area, highscatter peak intensity area (R4) were condensed and fragmented, and hada morphology consistent with cells in the late stages of apoptosis.Backgating PE⁺ cells (identified in the histogram shown in FIG. 4E) inyellow verified that R3 gated cells were derived from the necrotictreatment group (FIG. 4F). This conclusion is further supported bymorphologic examination of cells in the image galleries of the R3 and R4gated cells, and confirms that the low area/high texture cells wereapoptotic (HLA-class I PE⁺ cells containing fragmented 7-AAD stainingnuclei; lower right gallery) while high area/low texture cells werenecrotic (HLA class I-PE⁺ cells containing uniform 7-AAD stainingnuclei; upper right gallery). Thus, the data obtained from multispectralimaging is provided in a form that allows one to distinguish similarlystained cells, such as late apoptotic cells from necrotic cells.

Example 11 Complex Morphologic Feature Identity Using MultispectralImaging

Multispectral image data collection not only enables calculation ofstandard intensity-based parameters and statistics employed inconventional flow cytometry, but also permits quantitation of numerousother morphologic features (e.g., cell area, perimeter, aspect ratio,texture, spot counts, cell centroid, gradient intensity, spatialfrequency). Using this capability, it is possible to distinguish allfour cell populations (i.e., live, early apoptotic, late apoptotic andnecrotic cells) in a single step using morphologic features derived from7-AAD, brightfield and darkfield imagery (and in the absence of otherstaining procedures often used) to “identify” apoptotic cells.

By subtracting the 7-AAD image area (nuclear size) from the Brightfieldarea (cell size), a value is obtained that is an indication ofcytoplasmic size. When this complex morphologic feature (herein referredto as “Brightfield 7-AAD Area”) was used in conjunction with a featurederived from darkfield imagery (i.e., 488 nm spatial scatter frequency,which is an indication of internal cell complexity or cell granularity),four subpopulations of cells became evident (see FIG. 5). The 488 nmspatial scatter frequency can be calculated by computing the standarddeviation of the individual pixel intensities within the segmented darkfield image mask.

Live cells (depicted in blue, FIG. 5, center panel) excluded thecell-impermeant 7-AAD fluorescent DNA binding dye, which minimized thenuclear image area and resulted in cells with a large calculatedcytoplasmic area.

Early apoptotic cells (depicted in green, FIG. 5, center panel) are justas effective as live cells at excluding 7-AAD, but their totalbrightfield area is slightly smaller due to the early stages ofcytoplasmic blebbing, thereby resulting in an intermediate value for the“Brightfield-7AAD Area” parameter. Also associated with the early stagesof apoptosis is a significant increase in 488 nm scatter peak intensity,which clearly separates these cells from live cells on the vertical axisof the dot-plot.

Necrotic cells (depicted in yellow, FIG. 5, center panel) and lateapoptotic cells (depicted in red, FIG. 5, center panel) both hadcompromised membrane integrity, which permits free entry of 7-AAD andthus strong nuclear images of relatively large area, shifting thesepopulations to the left on the dot-plot. However, these two cellpopulations can be clearly separated based on the peak intensitymeasurements derived from their 488 nm scatter parameters. Necroticcells produce darkfield images of relatively low complexity compared tothe more complex and heterogeneous darkfield images of late apoptoticcells, thus clearly separating the two populations in the vertical axis.

Inspection of the associated image galleries associated with these fourgated populations of cells confirmed the classification of eachpopulation (see FIG. 5, upper and lower panels).

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

The invention in which an exclusive right is claimed is defined by thefollowing:
 1. A method for distinguishing late apoptotic cells fromnecrotic cells, comprising: based on images of a population of cells,identifying a group of cells that are either of a necrotic type or alate apoptotic type; and grouping cells in the population into a groupof late apoptotic cells and a group of necrotic cells based on abrightfield image area of the cells and a darkfield peak intensity areaof the cells, as determined from the images of the cells, where cellsare identified as being in the group of late apoptotic cells if thecells have both a low brightfield area and a high darkfield peakintensity, and as being in the group of necrotic cells if the cells haveboth a high brightfield area and a low darkfield peak intensity.
 2. Themethod of claim 1, wherein there is relative motion between the cellsand a detector used to produce images of the population of cells.
 3. Themethod of claim 1, wherein the cells in the group of late apoptoticcells are characterized as having a high texture in the brightfieldimage area, while the cells in the group of necrotic cells arecharacterized as having a low texture in the brightfield image area. 4.The method of claim 1, further comprising determining a spatialfrequency in the darkfield peak intensity area for the population ofcells, to further assist in grouping the cells in the population ofcells into either the group of late apoptotic cells or the group ofnecrotic cells.
 5. The method of claim 4, further comprising using thespatial frequency darkfield peak intensity area is an indicator ofinternal cell complexity or cell granularity of the cells in thepopulation of cells, wherein the cells in the group of necrotic cellsare characterized as having a relatively lower complexity compared tothe cells in the group of late apoptotic cells.
 6. The method of claim5, further comprising calculating the spatial frequency in the darkfieldpeak intensity area by computing a standard deviation of individualpixel intensities within a segmented dark field image mask for theimages of the population of cells.